DESCRIPTION: This is an application to continue studies on the physiology of cholesteryl ester hydrolases. The application has 4 specific aims. In the first aim, the cholesteryl esterase catalytic activity, mass and mRNA will be measured in liver and cultured hepatocytes from both normal and endocrinologically altered male and female rats and o the rats which have undergone a variety of nutritional, hormonal, or pharmacological interventions which affect cholesterol metabolism. Where changes are noted, transcription rates, mRNA turnover, protein turnover, and levels of enzyme phosphorylation will all be investigated. In the second specific aim, the gene for hepatic cholesteryl ester hydrolase will be characterized. The gene will be fully sequenced, the promoter sequence identified as well as potential regulatory sequences which will be done by homology to known regulatory elements in comparable enzymes. The gene structure and promoter characteristics will be compared with other enzymes of cholesterol metabolism and other carboxyl esterases. The third aim is to measure cholesterol ester hydrolase protein and mRNA in various regions of the liver as well as other cell types. This will be utilized as an indication of the function of the enzyme. Immunohistochemistry and in situ hybridization techniques will be utilized. In the fourth specific aim, adenovirus constructs encoding for the cDNA will be transfected into cultured hepatocytes as well as hamsters to evaluate the effects of overexpression or, in the case of specific anti sense sequences, the lack of enzymatic activity on cell viability, cholesterol levels, and the activity of other enzymes of cholesterol metabolism.